Localization of the CotY and ExsY proteins to the exosporium basal layer of Bacillus anthracis

Abstract Spores are an infectious form of the zoonotic bacterial pathogen, Bacillus anthracis. The outermost spore layer is the exosporium, comprised of a basal layer and an external glycoprotein nap layer. The major structural proteins of the inner basal layer are CotY (at the mother cell central pole or bottlecap) and ExsY around the rest of the spore. The basis for the cap or noncap specificity of the CotY and ExsY proteins is currently unknown. We investigated the role of sequence differences between these proteins in localization during exosporium assembly. We found that sequence differences were less important than the timing of expression of the respective genes in the positioning of these inner basal layer structural proteins. Fusion constructs with the fluorescent protein fused at the N‐terminus resulted in poor incorporation whereas fusions at the carboxy terminus of CotY or ExsY resulted in good incorporation. However, complementation studies revealed that fusion constructs, although accurate indicators of protein localization, were not fully functional. A model is presented that explains the localization patterns observed. Bacterial two‐hybrid studies in Escherichia coli hosts were used to examine protein–protein interactions with full‐length and truncated proteins. The N‐terminus amino acid sequences of ExsY and CotY appear to be recognized by spore proteins located in the spore interspace, consistent with interactions seen with ExsY and CotY with the interspace proteins CotE and CotO, known to be involved with exosporium attachment.


| INTRODUCTION
The genus Bacillus is comprised of soil-dwelling bacteria that utilize sporulation as a survival mechanism. When conditions are unfavorable for growth, such as nutrient limitation, the bacteria undergo a sporulation process to produce spores that are metabolically inert and resistant to a variety of environmental insults including heat and desiccation. The outer surface of the spore consists of glycoproteins. With Bacillus subtilis, this layer is referred to as the crust and is associated with the outer spore coat (Imamura et al., 2011;McKenney et al., 2010). Certain Bacillus species produce spores that possess an outer spore layer, the exosporium. It is a MicrobiologyOpen. 2022;11:e1327.
www.MicrobiologyOpen.com deformable protein shell that is separated from the spore coat by the interspace layer (Giorno et al., 2009). The exosporium consists of a basal layer and a hairlike nap layer containing the BclA collagen-like glycoprotein (Stewart, 2015;Sylvestre et al., 2002;Sylvestre et al., 2005). The basal layer of the B. anthracis exosporium is approximately 12-16 nm thick and appears to be comprised of two, approximately 5-nm-thick sublayers (Rodenburg et al., 2014). The exosporium is thought to be a semi-permeable barrier that excludes potentially harmful large molecules such as antibodies and hydrolytic enzymes, but permits the passage of small molecules such as germinants (Ball et al., 2008;Gerhardt & Black, 1961). The exosporium also confers hydrophobic properties on the spore, likely playing a role in persistence of spores in soil environments (Williams et al., 2013). However, with the zoonotic pathogen Bacillus anthracis, the exosporium is also the site of early interactions between the infectious spores and macrophages and dendritic cells of the host innate immune system during the initial stages of the infectious process Brahmbhatt et al., 2007;Oliva et al., 2009).
Synthesis of the exosporium initiates early in the sporulation process, at the stage of engulfment of the forespore by the mother cell. Before engulfment is complete, a thin layer, the bottlecap (or simply the cap) is evident at the mother cell central pole of the developing spore. Later in the sporulation process, coincident with spore coat assembly, the exosporium basal layer is assembled from the cap toward the noncap pole. Three important structural proteins of the basal layer are BxpB, CotY, and ExsY (Boydston et al., 2006;Johnson et al., 2006;Lablaine et al., 2021;Steichen et al., 2005;Sylvestre et al., 2005;Terry et al., 2017). B. anthracis mutants deleted for the exsY determinant produce spores possessing only the bottlecap portion of the exosporium (Boydston et al., 2006). This corresponds to approximately 25% of the exosporium at the mother cell central pole of the developing spore. The exsY mutants do not produce the noncap portion of the exosporium, indicating that ExsY is a major structural protein of the noncap basal layer. Mutants deleted for cotY produce an intact exosporium, but with altered assembly kinetics. The CotY protein is a structural component of the cap, and cotY mutants fail to produce the cap in the early stages of sporulation (Boone et al., 2018;Lablaine et al., 2021;Terry et al., 2017).
The exosporium is assembled at the time that the spore coat is assembled (Boone et al., 2018). During the early stages of assembly, the exosporium closely abuts the spore coat layer. Later, the exosporium separates from the spore coat, creating an electron translucent space between the two layers called the interspace (Giorno et al., 2009). During assembly, the exosporium is anchored to the spore coat. Mature spores from mutants lacking CotE or CotO proteins lack the exosporium layer (Boone et al., 2018;Giorno et al., 2007;Lablaine et al., 2021). CotE and cotO mutants produce the exosporium in sheets in the mother cell cytoplasm adjacent to the bottlecap pole of the spore (Boone et al., 2018;Giorno et al., 2007), indicating that assembly of the exosporium can occur in the absence of these anchoring proteins, but cannot encapsulate the developing spore and is lost following mother cell lysis. It is noteworthy that CotO has a role during crust assembly in B. subtilis, promoting spore encasement (Shuster et al., 2019). Mutant spores lacking the ExsA and ExsB proteins have also been reported to have exosporium attachment deficient phenotypes (Bailey-Smith et al., 2005;McPherson et al., 2010). However, loss of ExsA in Bacillus cereus also resulted in major defects in spore coat assembly, and the effects on exosporium attachment may be indirect effects due to loss of CotE or CotO.
This study is focused on the assembly of the CotY and ExsY basal layer proteins and factors which influence their correct localization within the exosporium. It also highlights the advantages, and limitations, of visualizing exosporium assembly utilizing fluorescent fusion proteins.

| DNA purification
The Wizard SV miniprep kit (Promega) was used to isolate plasmid DNA. For B. anthracis, the pellets from 5 ml cultures were frozen at −80°C overnight and thawed at 37°C before DNA extraction.
Genomic DNA was isolated using the Wizard Genomic DNA purification kit (Promega). For B. anthracis, the cell pellets were frozen at −80°C overnight and thawed at 37°C before DNA extraction.

| Construction of complementation and expression plasmids
Expression of Bacillus proteins was accomplished by the introduction of the gene with its native or heterologous promoter into the shuttle plasmids pMK4 (Sullivan et al., 1984) or pHPS2 (Thompson et al., 2011). The shuttle plasmid pHPS2 is a derivative of pHP13, it is a relatively low copy number replicon with a copy number of~5 in B. subtilis hosts (Haima et al., 1987). The cotY and exsY chimeras as well as the promoter exchanged constructs were constructed by splicing using overlapping extension polymerase chain reaction (PCR) (Horton et al., 1993

| Immunolabeling of spores
Ten milligrams of spores were resuspended in 750 μl SuperBlock Blocking buffer (Thermo Scientific) and incubated for at least 20 min at room temperature. The spores were then harvested by centrifugation and the spore pellet was resuspended in 250 μl SuperBlock blocking buffer with 1 μl primary antibody and incubated at room temperature for 20 min (with mixing every 5 min). Rabbit polyclonal anti-rBclA antibodies were used (Thompson et al., 2007). Following incubation with the primary antibody, the spores were harvested by centrifugation and washed with 750 μl of SuperBlock blocking buffer.
The pellet was then resuspended in 250 μl of SuperBlock Blocking buffer with secondary antibody conjugate (1:250 goat anti-rabbit IgG-Alexa Fluor 568; Invitrogen). The spores were incubated at room temperature for 20 min, pelleted, and washed with 750 μl SuperBlock blocking buffer, followed by three washes with 750 μl PBS, and finally resuspended in 250 μl PBS. The spores were examined by epifluorescence microscopy using a Nikon E600 epifluorescence microscope using the mCherry filter set.

| Protein interaction analysis by the bacterial two-hybrid method
The procedure of Karimova et al. (2017) was used. Plasmids pKT25, pKNT25, pUT18, and pUT18C were utilized, thus obtaining hybrid proteins with the T18 or T25 domains of adenylate cyclase on their N-or C-terminus. All plasmid constructs (Table A1)   ExsY-mCherry fusions labeled~75% of the spore surface with a paucity of fluorescence at one pole (a noncap region distribution pattern) (Figure 1). Spores from Sterne cells harboring compatible plasmids encoding CotY-mCherry and ExsY-eGFP produced spores with CotY-mCherry present at one pole and ExsY-eGFP predominantly at the noncap portion of the exosporium (Figure 1b). Table 1 provides a summary of the findings from the spore images shown in

| The ExsY-mCherry fusion protein is not fully functional in basal layer assembly
Spores from an exsY deletion mutant produce only the exosporium cap region (Boydston et al., 2006;Johnson et al., 2006). Expression  3.4 | The N-terminal sequences of CotY and ExsY do not fully account for the differential localization patterns of the two proteins The fusion protein localizes to the exosporium cap site, but the fluorescence was below the limit of detection in mature spores. (q) ΔexsY pHPS2-P cotY -cotY-mcherry; (r) ΔexsY pHPS2-P cotY -cotY NT-mcherry; (s) ΔexsY pHPS2-P cotY -cotY CT-mcherry. The white arrows in (g) indicate positions of spores where the fusion protein was positioned around the entire spore periphery, rather than at one pole. The yellow arrows in (r) point out the stronger fluorescence at the cap, but faint fluorescence is evident in the noncap portion of the exosporium.

| exsY and cotY single-copy expression patterns
spores (Figure 10b,c). CotY-eGFP localized at the cap pole while ExsY-mCherry was found at the noncap portion of the exosporium.

| CotY and ExsY protein interactions in a bacterial two-hybrid system
To identify potential interactions between the CotY and ExsY proteins and their truncated derivatives, we used a bacterial twohybrid system based on adenylate cyclase from Bordetella pertussis. His-tagged proteins were reported to self-assemble into sheets in the cytoplasm of the E. coli hosts (Terry et al., 2017). We found that the B. anthracis ExsY protein could productively pair with itself, regardless of whether the adenylate cyclase domain (AC) was fused to the N-terminus or the C-terminus of the protein (Figure 11a). The pairing was also observed with one ExsY partner having the AC at the N-terminus and the other having the AC at the C-terminus. We examined potential interactions with the truncated derivatives of ExsY and CotY (Figure 11b,c). The 29-residue ExsY N-terminus sequence gave no evidence of partnering with fulllength ExsY, the C-terminus protein, or with itself. The 123 residue C-terminus ExsY sequence was found to be capable of partnering with the ExsY full-length protein, although the magnitude of the β-galactosidase response was variable with the AC domains at the C-terminus of the protein, depending on which AC domain was attached to the truncated ExsY protein ( Figure 11b).
With the N-terminus AC fusions, strong activity was obtained with all of the combinations of ExsY with ExsY CT (Figure 11c). The ExsY CT protein was, however, not capable of self-association in this assay. The CotY 119 residue CT protein was not capable of partnering with full-length CotY or with itself with either the N-terminus or C-terminus AC fusions. The failure to associate with full-length CotY was surprising given the results obtained with ExsY interactions with its CT form and the sequence similarity of the CotY and ExsY CT protein forms. The results were consistent with the CotY CT-mCherry fusion displaying inefficient incorporation into the exosporium in the cotY null mutant spores ( Figure 6).

| CotY and ExsY protein interactions with CotE and CotO in a bacterial two-hybrid system
During exosporium assembly, the basal layer is anchored to the spore coat by a linkage system that involves the CotE and CotO proteins (Boone et al., 2018;Giorno et al., 2009). Mutants lacking either of these proteins produce an exosporium that fails to associate with the spore surface and is found as sheets at the mother cell central pole of the developing spore. N-terminus His 6 -tagged recombinant CotE and CotO proteins were found to interact, with the CotE protein thought to be at the outer spore coat and CotO further toward the exosporium basal layer (Boone et al., 2018).  Timing of expression appears to be the most important feature that results in CotY appearing predominantly at the bottlecap region of the exosporium basal layer and the later expressed ExsY protein predominantly positioned in the noncap portion of the exosporium.
Transcription of the B. thuringiensis exsY gene occurs with the σ Kbearing RNA polymerase and given the sequence identities, this is likely true with B. anthracis (Peng et al., 2016). Transcription of cotY has not been studied, but the promoter has a good match to the σ K consensus sequence. Potentially active transcription factors that may impact transcription kinetics have not yet been investigated.
Promoters recognized by σ K and the earlier acting σ E have almost identical −10 consensus sequences with the principal difference between the two classes of promoters residing at their −35 regions (Eichenberger et al., 2003). Therefore, some promoters under the control of σ K might also be recognized to some extent by σ E . If this is true for the cotY promoter, it may explain the earlier initiation of transcription. In prior TEM studies, the cap is the earliest appearing exosporium structure, first evident during the engulfment stage of sporulation (Boone et al., 2018). At this early stage of sporulation, the cap is immature, lacking the electron density and nap layer characteristic of the exosporium of mature spores. CotY, whose gene is transcribed earlier than that of exsY, is the major structural component of the cap. CotY is positioned at the mother cell central pole of the spore through interactions with a connector chain with the spore coat. CotE and CotO are known to be components of this linkage structure (Boone et al., 2018;Lablaine et al., 2021 noncap self-assembly. Excess (unincorporated) ExsY and/or CotY proteins would be lost following mother cell lysis.
After the cap forms, noncap assembly initiates and ExsY is incorporated. With mutants lacking ExsY, only the cap structure is formed (Boydston et al., 2006). Extension of the cap into the noncap portion of the spore is likely limited by the amount of CotY produced, as transcript levels of cotY decline in the later stages of spore maturation (Bergman et al., 2006 as plotted in Figure 7) Through the elegant studies of Terry et al. (2017), it was determined that ExsY, and to a lesser extent CotY, can self-assemble.
ExsY forms a two-dimensional lattice that is stabilized by disulfide linkages and electron crystallography analysis indicating the presence of the same overall structural features of an intact exosporium. For self-assembly to occur, the protein monomers must be able to recognize other subunits, be correctly positioned, and ultimately stabilized by disulfide bond formation. Our studies with ExsY-mCherry indicated that this fusion protein can be incorporated into the exosporium basal layer, but cannot serve as a substrate for subsequent monomer incorporation. The mCherry-ExsY protein, however, fails to incorporate at detectable levels in the presence of ExsY and poorly incorporates in the absence of the unfused protein.
This suggests a defect in the addition of the N-terminus fusion protein to a growing ExsY sheet, possibly through steric hindrance.
Surprisingly, the AC fusions at the N-terminus of both ExsY and CotY did not weaken or prevent protein-protein interactions in the bacterial two-hybrid assays. It is possible that although the three proteins fused (mCherry, AC T25, and AC T18) are not substantially different in mass (26,722, 18,951, and 24,017 Da, respectively), differences in secondary structure could impact steric effects.
Alternatively, the putative steric effect may not be on the capacity of the fusion protein to initially interact but prevents the proper alignment needed for the formation of the stabilizing disulfide bonds. This latter step would not be needed for adenylate cyclase activity in E. coli but would be required for the proper positional fixing of the protein in the exosporium sheet structure.
The short CotY and ExsY N-terminus sequences were sufficient to position the mCherry reporter around the spore, even in mutants that produce only the cap portion of the exosporium or lack the exosporium entirely. This interaction likely results from interactions with proteins within the interspace layer of the spore. The NT proteins do not measurably interact with the CotY or ExsY proteins.
On the other hand, the absence of the NT sequences from the ExsY CT protein did not disrupt interactions with ExsY or CotY, although ExsY CT self-interactions did not demonstrably occur. Thus, ExsY selfassembly requires the NT sequences. Despite the high sequence identity of the CotY CT and ExsY CT proteins, the CotY CT protein failed to interact with the full-length CotY protein, unlike the situation observed with ExsY CT/ExsY pairs. CotY CT-mCherry, however, can interact with full-length CotY protein, as evident by the cap labeling in the exsY null mutant spores. This labeling, however, was relatively weak, suggestive of weaker interactions.
In our model of exosporium assembly (Figure 13 pairing that leads to disulfide bond formation and thus stable incorporation. However, the interactions must be weak, given that the ExsY-mCherry protein was shown to assemble around the spore whereas the mCherry-ExsY protein remained in the cytoplasm of late-stage sporulating cells (Figure 4). With Sterne, the unfused CotY and ExsY proteins thus outcompete the fusion proteins for incorporation. The mCherry-ExsY fusion protein, expressed in the exsY null host, has no ExsY to compete with, which results in labeling at the CotY-containing bottlecap margin.
Following synthesis of the basal layer, the size of the interspace increases, possibly due to cleavage of the CotE/CotO chain that positioned the exosporium during the assembly process (Boone et al., 2018). Possible evidence for such a proteolytic event was the finding of an N-terminal CotE peptide (residues 2-14) in an exosporium proteomic study (Todd et al., 2003).